scholarly journals Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

2010 ◽  
Vol 189 (7) ◽  
pp. 1079-1086 ◽  
Author(s):  
Jayati Sengupta ◽  
Cyril Bussiere ◽  
Jesper Pallesen ◽  
Matthew West ◽  
Arlen W. Johnson ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Structural Data ◽  
Release Mechanism ◽  
Nuclear Pore ◽  
Subunit Joining

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

scholarly journals Reengineering Ribosome Export

2009 ◽  
Vol 20 (5) ◽  
pp. 1545-1554 ◽  
Author(s):  
Kai-Yin Lo ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Large Subunit ◽  
Nuclear Export Signal ◽  
Ribosomal Subunit ◽  
Nuclear Pore ◽  
Multiple Receptors ◽  
Ribosomal Subunit Protein ◽  
Specific Receptors ◽  
Ribosome Export ◽  
Export Receptors

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.

scholarly journals RanBP2/Nup358 Provides a Major Binding Site for NXF1-p15 Dimers at the Nuclear Pore Complex and Functions in Nuclear mRNA Export

2004 ◽  
Vol 24 (3) ◽  
pp. 1155-1167 ◽  
Author(s):  
Daniel Forler ◽  
Gwénaël Rabut ◽  
Francesca D. Ciccarelli ◽  
Andrea Herold ◽  
Thomas Köcher ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Mrna Export ◽  
Stable Complex ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cytoplasmic Filaments ◽  
Nuclear Levels ◽  
Pore Complexes

ABSTRACT Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.

scholarly journals The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

1999 ◽  
Vol 10 (3) ◽  
pp. 649-664 ◽  
Author(s):  
Katharine S. Ullman ◽  
Sundeep Shah ◽  
Maureen A. Powers ◽  
Douglass J. Forbes
Keyword(s):  
Nuclear Export ◽  
Critical Role ◽  
Adaptor Protein ◽  
Nucleocytoplasmic Transport ◽  
Protein Export ◽  
Nuclear Pore ◽  
Physical Interaction ◽  
Receptor Complexes ◽  
Importin Α ◽  
Rna Export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin β to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin α/β or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

Structure of the translating Neurospora ribosome arrested by cycloheximide

2021 ◽  
Vol 118 (48) ◽  
pp. e2111862118
Author(s):  
Lunda Shen ◽  
Zhaoming Su ◽  
Kailu Yang ◽  
Cheng Wu ◽  
Thomas Becker ◽  
...  
Keyword(s):  
Binding Site ◽  
Messenger Rna ◽  
Large Subunit ◽  
Structural Data ◽  
Binding Pocket ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Translation Elongation ◽  
Elongation Step ◽  
A Site

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX’s mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

scholarly journals Joining the interface: a site for Nmd3 association on 60S ribosome subunits

2010 ◽  
Vol 189 (7) ◽  
pp. 1071-1073 ◽  
Author(s):  
Marlene Oeffinger
Keyword(s):  
Binding Site ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Adaptor Protein ◽  
Cryoelectron Microscopy ◽  
Structural Description ◽  
Ribosomal Subunits ◽  
Cell Biol ◽  
Conformational Model ◽  
A Site

The adaptor protein Nmd3 is required for Crm1-dependent export of large ribosomal subunits from the nucleus. In this issue, Sengupta et al. (2010. J. Cell Biol. doi:10.1083/jcb.201001124) identify a binding site for yeast Nmd3 on 60S ribosomal subunits using cryoelectron microscopy and suggest a conformational model for its release in the cytoplasm. The study provides the first detailed structural description of a ribosome biogenesis factor in complex with the large subunit.

scholarly journals Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Ilyas Chachoua ◽  
Ilias Tzelepis ◽  
Hao Dai ◽  
Jia Pei Lim ◽  
Anna Lewandowska-Ronnegren ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Binding Site ◽  
Nuclear Export ◽  
Nuclear Architecture ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Nuclear Pore ◽  
Super Enhancer ◽  
Hct 116 Cells ◽  
Canonical Wnt

AbstractAbnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer-(OSE)-mediated gating of the active MYC to the nuclear pore in a poorly understood process. We show here that the principal tenet of the WNT-regulated MYC gating, facilitating nuclear export of the MYC mRNA, is regulated by a CTCF binding site (CTCFBS) within the OSE to confer growth advantage in HCT-116 cells. To achieve this, the CTCFBS directs the WNT-dependent trafficking of the OSE to the nuclear pore from intra-nucleoplasmic positions in a stepwise manner. Once the OSE reaches a peripheral position, which is triggered by a CTCFBS-mediated CCAT1 eRNA activation, its final stretch (≤0.7 μm) to the nuclear pore requires the recruitment of AHCTF1, a key nucleoporin, to the CTCFBS. Thus, a WNT/ß-catenin-AHCTF1-CTCF-eRNA circuit enables the OSE to promote pathological cell growth by coordinating the trafficking of the active MYC gene within the 3D nuclear architecture.

scholarly journals Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

2000 ◽  
Vol 151 (5) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jennifer Hei-Ngam Ho ◽  
George Kallstrom ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Adapter Protein ◽  
Leptomycin B ◽  
Ribosomal Subunits ◽  
Late Step

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.

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